1. Field of the Invention
This invention pertains to a method for transforming and regenerating soybeans. Specifically, a method of transforming and regenerating soybeans, using soybean hypocotyl explants, and either bacteria-mediated or biolistic methods (micro-particle bombardment) to effect genetic transformation of a large variety of cultivars, coupled with plant regeneration from the hypocotyls, provides a reliable means for introducing genetic variation.
2. Background of the Prior Art
Soybean [Glycine max (L.) Merr.] is one of the world's most important agronomic crops. Between 120 and 130 million acres are planted annually, resulting in 105 million tons of seed. Soybeans have dominated world oilseed production among the eight major oilseeds traded in international markets, accounting for over 97% of all world oilseed production since 1965. The value of the crop is estimated to be over 20 billion dollars. Both soybean oil and protein are used extensively in food products for human consumption. In the United States, 5% of the total protein is derived from grain legumes and up to 65% of the oil used by the food processing industry comes from soybean (Hoskin, USDA Econ. Res. 1-35 (1987), Smith and Huyser, 1-22 (1987).
A great deal of effort has been made towards the development of new cultivars of soybean with improved disease, pest, herbicide resistance along with increased nutritional value. However traditional breeding programs have been restricted because soybean germplasm is extremely narrow and the majority of the soybean cultivars in use are derived from very few parental lines Christou et al., TIBTECH 8:145-151 (1990).
The development of gene transformation techniques has created an alternate path to the genetic improvement of many crops such as increased disease, pest and herbicide resistance, as well as quality improvement. However, success with these approaches has been limited in soybean due to various in vitro difficulties attributed to soybean Christou, supra. The design of genetic engineering protocols for soybean would include the development of an efficient plant regeneration system.
The majority of reports on soybean regeneration utilized cotyledons from immature zygotic embryos induced to undergo somatic embryogenesis Liu et al., In Vitro Cell. Dev. Biol. 28P:153-160 (1992), which could entail protracted culture periods. Soybean regeneration through short meristem cultures resulted in up to 35% explants responding to plant regeneration 4 weeks after culture Kartha et al., Can. J. Bot., 59:1671-1679 (1981). Regeneration via organogenesis utilizing different explants has been reported with a maximum of 97% of explants responding and 3 shoots produced per explant 10 weeks after culture, and 38% of shoots developing roots for another 4 weeks Yeh et al., J. Argric. Assoc. China, 40:77-90 (1991). However, interactions between genotype and in vitro cultural conditions (medium, explant and light treatment) have not been reported in regeneration via organogenesis or meristem culture in soybean, although it has been studied in regeneration via somatic embryogenesis and was proven important Powell et al., Heredity 58:75-801 (1987); Komatsuda et al., Crop. Sci. 31:333-337 (1991).
U.S. Pat. No. 5,322,783, Tomes et al., is directed to a method for transformation of soybean tissue which calls for treating cotyledonary node cells with a cytokinin, and then bombarding the cells with microparticles carrying specific vectors and exgenogeous DNA. U.S. Pat. Nos. 5,169,770 and 5,376,543, Chee et al., focus on a different method of transforming soybeans, to produce transgenic plants. In the process described in these patents, seeds are germinated, and the meristematic or mesocotyl cell tissues are inoculated with bacterial cells, specifically Agrobacterium strains, which through infection, transfers DNA into these explants. Transgenic plants could ensue provided this transfection transformation was successful and occured prior to differentiation.
Any plant transformation program also requires a regeneration program. In U.S. Pat. No. 4,992,375, Wright et al., a process is described which calls for excising the cotyledonary node region from a donor plant, and culturing the explant in a nutrient media containing cytokinin, until shoots arose from resultant callus. The shoots are then rooted. U.S. Pat. No. 5,416,011, Hinchee et al., also utilizes a cotyledon explant, which requires removal of the hypocotyl, saving and separating the cotyledons and inserting a chimeric gene by inoculation with Agrobacterium tumefaciens vectors containing the desired gene. This reference, and many others, employ the GUS histochemical marker to determine successful transformation.
Generally, the processes developed are categorized by a variety of inadequacies. Reliable transformation and regeneration is not accomplished. The formation of shoots, and eventual rooting, takes place only in a tiny fraction of the cases. Further, successful transformation and successful regeneration are frequently cultivar-specific, with no broad success. Among investigators reporting random, and overall poor success, are Wayne et al., Plant Mol. Biol. (1988); Finer et al., In Vitro Cell. Dev. Biol. (1991); Sato et al., Plant Cell Reports (1993); Moore et al., Plant Cells Reports (1994); Parrott et al., In Vitro Cell. Dev. Biol. (1994) and Stewart et al., Plant Physiol. (1996). While limited successes in producing transgenic plants are reported (e.g., 1 out of 195 in Parrott et al. 1994), success is random, and not predictable.
Accordingly, it remains an object of those of skill in the art to develop a reliable, repeatable and non-cultivar-limited method for transforming and regenerating soybeans.